Action of calpastatin in prevention of cardiac L-type Ca2+ channel run-down cannot be mimicked by synthetic calpain inhibitors

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Abstract
Activity of L-type Ca2+ channels in a membrane patch disappears rapidly when the patch is excised from the cell into an artificial solution. This channel run-down observed in isolated membrane patches can however, be prevented by application of calpastatin, an endogenous protease inhibitor, and ATP. The high specificity of calpastatin for the protease calpain would clearly point to a participation of calpain activity in the run-down of Ca2+ channels. In an attempt to examine a possible involvement of calpain, three synthetic and rather specific calpain inhibitors were substituted for calpastatin. One of these inhibitors chosen for its membrane permeability in addition allowed calpain activity to be inhibited even before patch excision. The potency of these compounds in inhibiting calpain, specificallyμ- and m-calpain, was first determined in a biochemical assay and then compared with their efficacy in preventing Ca2+ channel run-down. Surprisingly, calpastatin was least effective in calpain inhibition but by far the most potent in prevention of Ca2+ channel run-down. In addition run-down of Ca2+ channel activity was examined for its reversibility, which would not be expected upon involvement of a proteolytic process. However, Ca2+ channel activity clearly recovered after run-down by application of calpastatin. In contrast, synthetic calpain inhibitors were unable to reverse Ca2+ channel run-down. These results indicate that proteolysis might only be partially responsible for channel run-down and suggest an as yet unidentified function for calpastatin beyond its inhibitory action on calpain in the regulation of Ca2+ channel activity.